T lymphocytes play a critical role in mediating and regulating immune responses. Constantly surveying the body, T cells must be able to distinguish self from non-self. Although T cell development in the thymus plays a critical role in "teaching" T cells to distinguish between self and non self, peripheral mechanisms also play an important role. One of the mechanisms is co-stimulation. T cells are thought to require two signals to become activated. Engagement of the TCR without the second "co-stimulatory" signal leads to T cell unresponsiveness. Extensive studies over the last five years, however, have yet to reveal the biochemical nature of the co-stimulatory signal. Here, the functions of CD28 and CD2 as co-stimulatory molecules will be analyzed. One notable feature of the cytoplasmic domains of CD28 and CD2 is the presence of proline-rich sequences which could serve as ligands for SH3 domains. The recently solved crystal structures of the Src- kinases, lck and hck, suggest that an important mechanism for src-kinase activation will be via SH3 engagement. Preliminary data demonstrate that peptides based on the sequences of CD28 and CD2 can serve as potent activators of lck, fyn and ITK. This appears to be specific as distinct peptides activate different kinases. In specific aim #1, we will determine the structural basis for this effect. In specific aim #2, we propose to delineate critical structural features of CD28 and CD2 in primary T cells. It is still not known which sequences of CD28 and CD2 are critical for its ability to regulate T cell anergy. All of the CD28 structure-function studies to date were performed in transformed T cells which cannot be anergized. Experiments are proposed using retroviral and transgenic approaches to reconstitute CD2/CD28 knockout animals with wild-type and mutated forms of CD28 and CD2. Lastly, it was recently proposed that fyn activation of the small G- protein, Rap1, is responsible for the maintenance of T cell unresponsiveness. Experiments (specific aim #3) are planned to test this hypothesis using transgenic mice expressing B-Ray, and mutated forms of Rap1. These experiments are designed to definitively test the role of Rap1 in the process of anergy.